Philippe Pognonec

A novel function for fragile X mental retardation protein in translational activation.

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

The Ets2 Transcription Factor Inhibits Apoptosis Induced by Colony-Stimulating Factor 1 Deprivation of Macrophages through a Bcl-xL-Dependent Mechanism

ABSTRACT Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-xpromoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate thebcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether thebcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression ofets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, andbcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription.

cDNA cloning and characterization of the transcriptional activities of the hamster peroxisome proliferator-activated receptor haPPAR gamma

We have isolated a cDNA corresponding to the hamster peroxisome proliferator-activated receptor haPPAR gamma, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR gamma mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR gamma may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal beta-oxidation. haPPAR gamma binds efficiently in the presence of retinoid X receptor alpha (RXR alpha) to a peroxisome proliferator response element (PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the rate-limiting enzyme of peroxisomal beta-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR gamma/RXR alpha on the PPRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR alpha, we show that the transcriptional activities of mPPAR and haPPAR gamma are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR.

Development of a new bicistronic retroviral vector with strong IRES activity

BackgroundInternal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in todays cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies.ResultsWe observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function.ConclusionFrom these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP).

Bcl-xL Expression Correlates with Primary Macrophage Differentiation, Activation of Functional Competence, and Survival and Results from Synergistic Transcriptional Activation by Ets2 and PU.1

Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-xL protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression ofbcl-x L , ets2, andPU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate thebcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-xL in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages.

ERK and cell death: cadmium toxicity, sustained ERK activation and cell death

Extracellular signal‐related kinase (ERK) is a key player in cell signaling. After 25 years of investigation, ERK has been associated with every major aspect of cell physiology. Cell proliferation, cell transformation, protection against apoptosis, among others, are influenced by ERK function. Surprisingly, ERK has also been associated with two apparently opposing processes. The involvement of ERK in cell proliferation has been extensively described, as well as its function in postmitotic cells undergoing differentiation. The analysis of these apparent discrepancies has led to a more precise understanding of the multiple functions and regulations of ERK. More recently, several groups have identified a new and unexpected role for ERK. Although being accepted as an important player in the protection against cell death by apoptosis, it is now clear that ERK can also be directly linked to cell death signaling. Here, we review the role of ERK in cell response to cadmium and its association with cell toxicity. In this system, ERK is subjected to a continuous activation that can last for days, which ultimately results in cell death. Cadmium entry into cells is responsible for this sustained ERK activation, probably via reactive oxygen species production, and protein kinase C has a negative action on this cadmium‐dependent ERK activation by modulating cadmium entry into cells.

Constitutive c-ets2 expression in M1D+ myeloblast leukemic cells induces their differentiation to macrophages.

The expression of c-ets2 is rapidly induced in a variety of myelomonocytic cell lines as they differentiate into macrophages. We find that constitutive expression of c-ets2 in the M1D+ myeloblast leukemic cell line (M1ets2) is sufficient to push these cells to a more differentiated state. The expression of several differentiation-specific genes is upregulated in M1ets2 cells, including those encoding macrophage-specific lysozyme M and tumor necrosis factor alpha, which are involved in bacteriolytic and inflammatory processes, respectively. Transcription factors c-jun and junB, previously shown to induce partial macrophage differentiation when overexpressed in myelomonocytic leukemia cell lines, are also upregulated in M1ets2 cells. The upregulation of junB is the result of a direct interaction of Ets2 with ets binding sites of the junB promoter, since transient or constitutive Ets2 expression in M1D+ cells activates junB transcription via ets binding sites. In addition, transfection of a dominant negative mutant of Ets2, devoid of its transcriptional activation domain, greatly reduces transcriptional activities of the junB promoter in M1ets2 cells. Finally, unlike their parental M1D+ counterparts, M1ets2 cells secrete the macrophage colony-stimulating factor, CSF-1, and are able to phagocytize. Taken together, these results show that when the immature myeloid M1D+ cell line constitutively expresses c-ets2, these cells acquire different functions of mature macrophages.

Development of an inducible suicide gene system based on human caspase 8

Suicide gene-therapy strategies are promising approaches in treating various diseases such as cancers, atherosclerosis, and graft-versus-host-disease. Here, we describe the development of a new effector gene based on inducing functional caspase 8, the initiator caspase in the death-receptor pathway. We constructed vectors encoding a constitutively active form of human caspase 8 (CC8), and demonstrated the efficient killing of a variety of cell types in transfection and lentivirus-transduction assays. We then analyzed the ability to control the apoptotic activity of a caspase 8-derived construct through the ARIAD™ homodimerization system (FKC8), a system shown to be extremely effective in several cellular models upon retroviral and lentiviral gene transfer. Similarly, two transcription-regulation systems, muristerone-regulated and Tet-On, were tested to control the expression of CC8. The homodimerization-regulated system FKC8 was shown to be the most efficient system with low background activity in noninduced conditions. In the presence of a dimerizer, it was as active as the activated Tet-On system. From our data, we conclude that the dimerizer-dependent human caspase 8 represents a highly inducible and very powerful system to eradicate transduced cell populations. In addition to its application in experimental gene therapy, this variant may be highly useful for mechanistic research related to apoptosis.

The Role of the Ets2 Transcription Factor in the Proliferation, Maturation, and Survival of Mouse Thymocytes

In this study, we investigated the effects of Ets2 expression on the proliferation, maturation, and survival of thymocytes by establishing transgenic mice that specifically express Ets2 or a dominant negative form of Ets2, Δets2, in the thymus. We show that, in young animals, there are fewer T cells in Δets2 transgenic thymi and that the maturation of these T cells is affected at the CD4−CD8− double-negative to CD4+CD8+ double-positive transition compared with wild-type littermate mice. Partial recovery in the number of thymocytes and full T cell maturation are restored with increasing age of Δets2 transgenic animals. However, thymocytes from adult Δets2 transgenic mice cultured ex vivo are more sensitive to cell death and to glucocorticoid-induced apoptosis than are T cells from control littermate mice. We also show that T cells from adult ets2 transgenic mice proliferate faster than their wild-type littermates. The proliferation and survival of these T cells are clearly affected upon apoptotic signals: glucocorticoid-induced apoptosis induces T cells from ets2 transgenic mice to continue to proliferate in vivo and to survive better ex vivo than T cells from control littermates. It has been shown that c-Myc expression is required for thymic proliferation and improves thymocyte survival of dexamethasone-treated animals. We show that the expression of c-Myc, an Ets2 target, is elevated in T cells freshly isolated from thymi of ets2 transgenic mice pretreated with dexamethasone. Together, these results show that Ets2 plays a role in the proliferation and survival of thymocytes, implicating a Myc-dependent pathway.

Long-term extracellular signal-related kinase activation following cadmium intoxication is negatively regulated by a protein kinase C-dependent pathway affecting cadmium transport.

Extracellular signal‐related kinase (ERK) is a well‐known kinase taking part in a signal transduction cascade in response to extracellular stimuli. ERK is generally viewed as a kinase that is rapidly and transiently phosphorylated following mitogenic stimulation. This activation results in ERK phosphorylating further downstream targets, thus transmitting and amplifying the original stimulus, and ultimately resulting in the onset of cellular proliferation and/or protection against apoptosis. More recently, several groups have identified a strikingly new type of ERK activation that results in cell death. This activation is very different from conventional ERK activation, as it occurs several hours after the original stimulation, and results in the sustained phosphorylation of ERK, which can be observed for up to several days. One way of inducing this delayed ERK activation is by low‐dose cadmium treatment. We show here that sustained ERK activation induced by cadmium in human kidney‐derived cells is inhibited following protein kinase C (PKC) activation, even when this activation occurs hours before intoxication. Furthermore, PKC inhibition results in an enhanced ERK activation in response to cadmium, even when inhibition is induced hours before intoxication. PKCε appears to be the most implicated isotype in this phenomenon. Finally, we present evidence suggesting that the ZIP8 transporter is involved in this process, as multiple small interfering RNAs against ZIP8 have a protective effect against cadmium treatment. Our results indicate that PKC activation negatively affects ZIP8 transporter activity, thus protecting cells against cadmium poisoning.